PROJECT SUMMARY Obesity and associated diseases, such as diabetes and hepatic steatosis, are characterized by excessive amounts of triglycerides (TGs) in tissues. To develop therapies for these diseases, an understanding of the molecular processes involved in the synthesis and storage of TGs is crucial. The final and only committed step in triglyceride synthesis is catalyzed by acyl CoA:diacylglycerol acyltransferase (DGAT) enzymes. Mammals possess two DGAT enzymes, DGAT1 and DGAT2, which differ substantially in their biochemical and physiological functions. Although we have learned much about these enzymes in recent years, may outstanding and important questions remain. The current proposal addresses these questions at the biochemical, cellular, and physiological levels by pursuing three specific aims. Aim 1 focuses on signaling pathways that regulate the activity of DGAT enzymes, focusing particularly on DGAT1 regulation during lipolysis. Aim 2 focuses on further elucidating physiological functions of DGAT1 by using murine genetic models with inactivation of the enzyme in specific tissues or cells relevant to fat metabolism, such as the adipose tissue, small intestine, skin, and macrophages. Aim 3 investigates an intriguing idea of manipulating DGAT expression in induced pluripotent stem (iPS) cells as a potential therapy for type 2 diabetes associated with obesity. Work resulting from these aims will advance the field of lipid synthesis as it relates to obesity. Because both DGAT enzymes are prospective targets of pharmaceutical approaches to treat obesity, diabetes, and hepatic steatosis, our studies also may have significant impact on the treatment of these diseases.